RS53157B - SCLEROSTINE ANTIBODIES - Google Patents

Abstract

An antibody or functional fragment thereof that specifically binds human sclerostin, wherein the antibody or functional fragment comprises six CDRs of the following amino acid sequences: HCDR1 sequences SEQ ID NO: 26, HCDR2 sequences SEQ ID NO: 27, HCDR3 sequences SEQ ID NO: 28, LCDR1 sequences SEQ ID NO: 29, LCDR2 sequences SEQ ID NO: 30 and LCDR3 sequences SEQID NO: 31. The application contains 7 more claims.

Description

Description of the invention

[0001] This invention is from the field of medicine, specifically from the field of antibodies to sclerostin. More specifically, the invention relates to high affinity antibodies that specifically bind human sclerostin and to the therapeutic use of such antibodies for various disorders or conditions in those humans that would benefit from an increase in at least one of the following: bone mass, bone mineral density, bone mineral content, or bone strength.

[0002] Osteoporosis is a disease in which the bone mineral density (BMD) is reduced, the microarchitecture of the bones is destroyed, and osteoporotic bones are at risk of breaking. Osteoporosis remains a major cause of long-term disability and mortality, particularly in the elderly. There are effective treatments for osteoporosis in the form of lifestyle modifications and pharmacotherapy, but available therapies are limited in number and effectiveness and are often associated with unwanted side effects and are not generally accepted by patients. A variety of antiresorptive agents, including calcitonin, bisphosphonates, estrogen replacement, and selective estrogen receptor modulators (SERMs) prevent further bone loss, but do not rebuild what is once lost. An anabolic agent that increases bone mass and bone mineral density and restores bone architecture is available in the form of human PTH(l-34). However, this therapeutic agent requires daily subcutaneous injection, often for a year or longer, leading to less patient compliance. |0003]Sclerostin, the product of the SOST gene, is intensively expressed in osteocytes in bones. Because of its role as a potent negative regulator of bone formation, sclerostin is a desirable target for therapeutic intervention in disorders or conditions that would benefit from an increase in at least one of the following: bone mass, bone mineral density, bone mineral content, or bone strength, eg, osteoporosis. Therefore, antibodies to sclerostin may prove useful as an anabolic approach to treating such disorders or conditions. PCT International Publication No. WO

2006/119107 discloses amino acid sequences of certain humanized antibodies to sclerostin in which all CDRs are murine, i.e., not changed to the CDRs of an antibody generated in a mouse. WO 2006/119062 discloses antibodies or fragments thereof capable of binding to sclerostin which are said to be useful in the treatment of a variety of bone-related conditions.

[0004] There is a need for an alternative sclerostin antibody that binds to human sclerostin with strong binding affinity and has a low IC50 value in the sclerostin bioactivity assay. Such an antibody can be predicted to have greater therapeutic value, particularly in osteoporosis, and to require lower dosing than a PTH(1-34) or sclerostin antibody with lower binding affinity (eg, higher KD) or lower IC50 value. Also, there is a need for an antibody specific for human sclerostin where the risk of an immune response to the antibody in the recipient of the antibody is reduced or the risk of instability is reduced, while the properties of the antibody having high binding affinity for human sclerostin and low IC50 values are maintained in the sclerostin bioactivity assay. The sclerostin antibodies of the present invention meet these needs and have the advantages associated therewith.

[0005] The antibodies of the present invention are chimeric or humanized monoclonal antibodies. They contain the specific polypeptide sequence provided herein, specifically bind human sclerostin with high binding affinity and can be used to increase at least one of the following: bone mass, bone mineral density, bone mineral content or bone strength in mammals, preferably in humans.

[0006] In one embodiment, the amino acid sequence of at least one CDR in the antibody of the present invention differs from the CDR at one position relative to the parent antibody, i.e., the antibody generated in the rodent (eg, mouse) from which the modified antibody, i.e., the humanized or chimeric antibody of the present invention was derived. for human sclerostin, a lower IC50 in the sclerostin bioactivity assay, relative to the parent antibody. An amino acid substitution in the CDR sequence of the present antibody relative to the parent antibody may result in a reduced immunogenic response to the antibody in the recipient. In addition, an amino acid substitution in the antibody CDR sequence of the present invention may reduce the risk of antibody instability, e.g., of asparagine with a different amino acid, reducing thus the risk of deamidation.

[0007] A first aspect of the present invention provides an antibody or functional fragment thereof that specifically binds to human sclerostin, wherein the antibody or functional fragment thereof contains six CDRs of the following amino acid sequences: HCDR1 with SEQ ID NO: 26, HCDR2 with SEQ ID NO: 27, HCDR3 with SEQ ID NO: 28, LCDR1 with SEQ ID NO: 29, LCDR2 with SEQ ID NO: 30 and LCDR3 with SEQ ID NO: 31.

[0008] Primarily, an antibody or a functional fragment thereof according to the present invention, contains a variable region of the heavy chain and a variable region of the light chain. The heavy chain variable region has the amino acid sequence of SEQ ID NO: 15 and the light chain variable region has the amino acid sequence of SEQ ID NO: 18.

[0009] Even better, the antibody or functional fragment thereof according to the present invention comprises a heavy chain polypeptide of the amino acid sequence SEQ ID NO: 3 and a light chain polypeptide of the amino acid sequence SEQ ID NO: 6.

[0010] Another aspect of the present invention provides a pharmaceutical composition comprising an antibody of the present invention and a pharmaceutically acceptable carrier or diluent.

[0011] Another aspect of the present invention provides for the administration of a drug an antibody or a functional fragment thereof according to the present invention.

[0012] Primarily, an antibody or functional fragment thereof according to the present invention is used to increase one of the following in a human: bone mass, bone mineral density, bone mineral content or bone strength and, more specifically, to treat diseases or disorders such as osteoporosis, osteopenia, osteoarthritis, pain related to osteoarthritis, rheumatoid arthritis, periodontal disease, or multiple myeloma in humans.

[0013] The antibody shown herein specifically binds human sclerostin and contains six CDR regions with amino acid sequences selected from the group consisting of (i) HCDR1 sequence SEQ ID NO: 20, HCDR2 sequence SEQ ID NO: 21, HCDR3 sequence SEQ ID NO: 22, LCDR1 sequence SEQ ID NO: 23, LCDR2 sequence SEQ ID NO: 24, and LCDR3 sequence SEQ ID NO: 25; (ii) HCDR1 sequence of SEQ ID NO: 26, HCDR2 sequence of SEQ ID NO: 27, HCDR3 sequence of SEQ ID NO: 28, LCDR1 sequence of SEQ ID NO: 29, LCDR2 sequence of SEQ ID NO: 30, and LCDR3 sequence of SEQ ID NO: 31; and (iii) HCDR1 sequence of SEQ ID NO: 32, HCDR2 sequence of SEQ ID NO: 33, HCDR3 sequence of SEQ ID NO: 34, LCDR1 sequence of SEQ ID NO: 35, LCDR2 sequence of SEQ ID NO: 36, and LCDR3 sequence of SEQ ID NO: 37.

[0014] In one example, the antibody specifically binds human sclerostin and contains a polypeptide of the heavy chain variable region ("HCVR", from the Eng. heavy chain variable region) and a polypeptide of the variable region of the light chain ("LCVR", from the Eng. light chain variable region) where (i) HCVR has an amino acid sequence of SEQ ID NO: 14, and LCVR has an amino acid sequence of SEQ ID NO: 17; (ii) HCVR has the amino acid sequence of SEQ ID NO: 15 and LCVR has the amino acid sequence of SEQ ID NO: 18; or (iii) HCVR has the amino acid sequence of SEQ ID NO: 16 and LCVR has the amino acid sequence of SEQ ID NO: 19.

[0015] In another example, the antibody specifically binds human sclerostin and comprises a heavy chain variable region polypeptide and a light chain variable region polypeptide where (i) the heavy chain polypeptide has the amino acid sequence of SEQ ID NO: 2 and the light chain polypeptide has the amino acid sequence of SEQ ID NO: 5; (ii) the heavy chain polypeptide has the amino acid sequence of SEQ ID NO: 3 and the light chain polypeptide has the amino acid sequence of SEQ ID NO: 6; or (iii) the heavy chain polypeptide has the amino acid sequence of SEQ ID NO: 4 and the light chain polypeptide has the amino acid sequence of SEQ ID NO: 7,

[0016] In one example, antibodies as defined herein, predominantly designated by SEQ ID number, are further characterized by having a binding affinity (Kd) for human sclerostin of about 10 pM or less, at 25°C. Good antibodies have macaque monkey sclerostin Kdza of about 100 pM or less at 25°C. Even better antibodies have a binding affinity for human sclerostin of about 10 pM or less, at 25°C, and a binding affinity for macaque monkey sclerostin of about 100 pM or less, at 25°C.

[0017] In another example, antibodies as defined herein, preferably designated by SEQ ID number, are further characterized by having an IC of 50 nM or less in a bone-specific alkaline phosphatase assay using human sclerostin. Preferably, these antibodies also have an ICD for human sclerostin of about 10 pM or less, at 25°C. More preferably, these antibodies have a human sclerostin Kdza of about 10 pM or less at 25°C and a macaque monkey sclerostin Kdza of about 100 pM or less at 25°C.

[0018] In another example, the present invention provides a pharmaceutical composition comprising an antibody of the present invention and a pharmaceutically acceptable carrier or diluent. Preferably, the pharmaceutical composition contains a homogeneous or substantially homogeneous population of the monoclonal antibody of the present invention and a pharmaceutically acceptable carrier or diluent.

[0019] The present invention includes the use of the antibody of the present invention for obtaining a drug. For use in a method for increasing at least one of the following: bone mass, bone mineral density, bone mineral content or bone strength in an animal, preferably a mammal, more preferably a human.

[0020] Also disclosed herein is a method of increasing at least one of the following: bone mass, bone mineral density, bone mineral content or bone strength comprising administering to a person in need thereof an effective amount of an antibody of the present invention.

[0021] Also disclosed herein is a method for treating a disease, condition, or disorder in a person that benefits from an increase in at least one of the following: bone mass, bone mineral density, bone mineral content, or bone strength, including, e.g., osteoporosis, osteopenia, osteoarthritis, pain related to osteoarthritis, periodontal disease, and multiple myeloma.

[0022] Here is further presented a method for detecting sclerostin protein in a biological sample, which consists in incubating an antibody of the present invention with a biological sample under conditions and for a time sufficient to allow the given antibody to bind to the sclerostin protein and to detect that binding. A suitable antibody for use in this detection assay has a heavy chain polypeptide of the sequence SEQ ID NO:40 and a light chain polypeptide of the sequence SEQ ID NO: 41; a heavy chain polypeptide of the sequence SEQ ID NO: 42, and a light chain polypeptide of the sequence SEQ ID NO: 43; heavy chain polypeptide of sequence SEQ ID NO: 2 and light chain polypeptide of sequence SEQ ID NO: 5; heavy chain polypeptide of sequence SEQ ID NO: 3 and light chain polypeptide of sequence SEQ ID NO: 6; or a heavy chain polypeptide of the sequence SEQ ID NO: 4 and a light chain polypeptide of the sequence SEQ ID NO: 7.

[0023] Also disclosed herein are isolated nucleic acid molecules that encode an antibody of the present invention, a vector containing that nucleic acid, optionally operably linked to control sequences recognized by a host cell transformed by that vector, a host cell containing that vector, a process for producing an antibody of the present invention that includes culturing the host cells so that the nucleic acid is expressed and, optionally, obtaining the antibody from the culture medium host cell.

[0024] The present invention provides antibodies that specifically bind human sclerostin, neutralize or antagonize at least one bioactivity of sclerostin in vitro or in vivo and further exhibit strong binding affinity for human sclerostin.

[0025] When the term "sclerostin" is used herein, it refers to the complete human protein, the amino acid sequences shown in SEQ ID NO: 1 or the mature form of the protein from which the signal sequences have been removed.

[0026] The term "antibody" when referring to the sclerostin antibody of the present invention (or, simply, "the antibody of the present invention"), as used herein, refers to a monoclonal antibody. "Monoclonal antibody" as used herein refers to a chimeric antibody or a humanized antibody, unless otherwise indicated. Monoclonal antibodies of the present invention can be obtained by, e.g., recombinant technologies, phage display technologies, synthetic technologies, e.g., CDR insertion, or combinations of these technologies or other technologies known in the art. "Monoclonal antibody" refers to an antibody derived from a single copy or clone, including, e.g., any eukaryotic, pokaryotic, or phage clone, and not the method by which they are obtained.

[0027] "Monoclonal antibody" or "antibody of this invention" or just "antibody" can be an intact antibody (containing the complete or entire Fc region), or a part or fragment of an antibody containing an antigen-binding part, for example, Fab fragment, Fab' fragment, or F(ab')2 fragment of a chimeric or humanized antibody. Particularly preferred antigen-binding fragments of the antibodies of the present invention retain the ability to inhibit or neutralize one or more bioactivities characteristic of mammalian sclerostinin in vivo or in vitro. For example, in one embodiment, the antigen-binding portion of the antibody of the present invention may inhibit the interaction of mature human sclerostin with one or more ligands and/or may inhibit one or more receptor-mediated functions of human sclerostin.

[0028] In addition, a "monoclonal antibody" or "antibody of the present invention" or just "antibody" as used herein, can be a single-chain Fv fragment that can be obtained by joining the linker sequence of DNA encoding LCVR and HCVR (See Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp 269-315, 1994). It is understood that, regardless of whether antigen-binding fragments or portions are specified, the term "antibody" as used herein includes such fragments or portions, as well as single-chain forms, unless otherwise indicated. As long as the protein retains the ability to specifically bind sclerostin it is covered by the term "antibody".

[0029] The term "specifically binds", as used herein, refers to a situation in which one member of a specific binding pair does not significantly bind to other molecules, except for its specific binding partner, as measured by a technique available in the art, e.g., a competitive ELISA assay, a BIACORE® assay, or a KINEXA® assay. This term is also applicable when, e.g., the antigen-binding domain of an antibody of the present invention is specific for a particular epitope carried by many antigens, in which case the antibody having the antigen-binding domain will be able to specifically bind to different antigens carrying that epitope. The term "epitope" refers to that part of a molecule that an antibody is able to recognize and bind with one or more of the antibody's antigen binding regions.

[0030] The term "bioactivity," as related to an antibody of the present invention, includes, but is not limited to, epitope or antigen binding affinity, the ability to neutralize or antagonize the bioactivity of sclerostin in vivo or in vitro, IC50 in a bone-specific alkaline phosphatase assay (e.g., as described herein in Example 2) or other in vitro assays for activity, in vivo and/or in vitro stability of the antibody, and immunogenic properties of the antibody, e.g., when to be given to a person. The aforementioned properties or characteristics can be observed or measured using techniques known in the art including, but not limited to, ELISA, competitive ELISA, surface plasmon resonance analysis, in vitro and in vivo neutralization assays, receptor binding, and immunohistochemistry on tissue sections from a variety of sources, including human, primate, or, as appropriate, any other source.

[0031] The term "bioactivity" related to sclerostin includes, but is not limited to, specific binding of sclerostin to another protein (e.g., a receptor or a member of the TGF-B family), one or more receptor-mediated functions performed by human sclerostin, signal transduction, immunogenic properties, in vivo or in vitro stability, effects on the activity levels of another protein in vivo or in vitro (see, e.g., Example 2), sclerostin expression levels, and distribution by tissues.

[0032] The term "inhibit" or "neutralize", as used herein in reference to the bioactivity of an antibody of the present invention, means the ability to substantially antagonize, prevent, limit, slow, destroy, eliminate, stop, reduce or reverse the bioactivity of sclerostin (eg, as measured herein in Example 2).

[0033] The term "Kabat numbering," as used herein, is recognized in the art and refers to a system of numbering amino acid residues that vary more (ie, are hypervariable) than other amino acid residues in the heavy and light chain regions of an antibody (Kabat, et al., Ann. NY Acad. Sci. 190:382-93 (1971); Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991). The positioning of the CDRs in the variable region of an antibody follows Kabat numbering or, simply, "Kabat".

[0034] A polynucleotide is "operably linked" when it is placed in a functional relationship with another polynucleotide. For example, a promoter or enhancer is operatively linked to a coding sequence if it affects the transcription of that sequence.

[0035] The terms "subject," "person," and "patient," used interchangeably herein, refer to mammals, primarily humans. In some embodiments, the subject is further characterized by a disease, disorder, or condition that would benefit from reduced sclerostin levels or reduced sclerostin bioactivity.

[0036] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is operably linked, and includes, but is not limited to, plasmid and viral vectors. Some vectors can autonomously replicate in the host cell, while other vectors can, after being introduced into the host cell, integrate into the host cell's genome and therefore replicate when the host genome is also replicated. Furthermore, some vectors can direct the expression of genes to which they are operably linked. Such vectors are called "recombinant expression vectors" (or just "expression vectors"). Exemplary vectors are well known in the art.

[0037] As used herein, the terms "cell", "host cell" and "cell culture" are used synonymously and include a single cell or cell culture that is the recipient of any isolated polynucleotide of the present invention or any recombinant vector containing a nucleotide sequence encoding an HCVR, LCVR or antibody of the present invention. A host cell includes cells transformed, transduced, or infected with one or more recombinant vectors or polynucleotides expressing the monoclonal antibody of the present invention or its heavy chain or light chain.

[0038] Each full-length antibody heavy chain contains an N-terminal heavy chain variable region (herein "HCVR") and a heavy chain constant region. Each full-length antibody light chain contains an N-terminal light chain variable region (herein "LCVR") and a light chain constant region. HCVR and LCVR regions can be further divided into regions of hypervariability, called complementarity determining regions ("CDRs"), between which there are more conservative regions, called framework regions ("FRs"). The functional ability of an antibody to bind a particular antigen or epitope is greatly influenced by the six CDRs in the variable region of the antibody. Each HCVR and LCVR consists of three CDRs (HCDR1, HCDR2 and HCDR3 in HCVR and LCDR1, LCDR2 and LCDR3 in LCVR) and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. CDRs contain most of the amino acids that form specific interactions with the antigen. The positioning of the CDR within the variable region is according to Kabat.

[0039] Light chains are classified as kappa or lambda and are characterized by a specific constant region as known in the art. Heavy chains are classified as gamma, mi, alpha, delta, or epsilon, and define the antibody isotype as IgG, IgM, IgA, IgD, and IgE, and several of these can be further subdivided into subclasses, eg, IgGj, IgG2, IgG3, IgG4. Each type of heavy chain is characterized by a specific constant region of sequence well known in the art. The kappa light chain constant region and the heavy chain constant regions of IgGi, IgG2, IgG3, IgG4 are preferred constant regions in the antibodies of the present invention. More preferably, the heavy chain constant region comprises a polypeptide of the amino acid sequence SEQ ID NO: 38 encoded by the polynucleotides of the sequence SEQ ID NO: 39. Chimeric antibodies may have constant regions of non-human origin, most preferably rat or mouse.

[0040] As used herein, "antigen-binding region" or "antigen-binding portion" refers to that part of an antibody molecule, within the variable region, that contains the amino acids that interact with the antigen and give the antibody its specificity and affinity for the antigen. This portion of the antibody includes the framework region amino acids necessary to maintain the proper conformation of antigen-binding amino acid residues.

[0041] A preferred antibody contains six CDRs of the amino acid sequence SEQ ID NOs: 20, 21, 22, 23, 24 and 25. Another preferred antibody contains six CDRs of the amino acid sequence SEQ ID NOs: 32, 33, 34, 35, 36 and 37. A more preferred antibody contains six CDRs of the amino acid sequence SEQ ID NOs: 26, 27, 28, 29, 30 and 31. The CDRs of these preferred antibodies are as set forth below in Table 1. The CDRs are positioned in the Kabat variable region.

[0042] A preferred antibody comprises the LCVR amino acid sequences of SEQ ID NO 17, 18 or 19. Other preferred monoclonal antibodies comprise the HCVR amino acid sequences of SEQ ID NO: 14, 15 or 16. Even more preferably the antibody comprises the LCVR sequences of SEQ ID NO: 17 and the HCVR sequences of SEQ ID NO: 14. An alternative antibody comprises the LCVR sequences of SEQ ID NO: 19 and HCVR. sequence of SEQ ID NO: 16. A more preferred antibody comprises the LCVR sequence of SEQ ID NO: 18 and the HCVR sequence of SEQ ID NO: 15. Such LCVRs are predominantly bound to the constant region of the light chain, primarily the cap chain. Such HCVRs are predominantly operably linked to a heavy chain constant region, primarily of human origin, generally IgGi or IgG4, most preferably to a heavy chain constant region of the amino acid sequence SEQ ID NO:38.

[0043] One preferred antibody contains a heavy chain polypeptide of the amino acid sequence SEQ ID NO: 2 and a light chain polypeptide of the amino acid sequence SEQ ID NO: 5. The heavy chain polypeptide of the amino acid sequence SEQ ID NO: 2 can be encoded, for example, by the polynucleotide sequence SEQ ID NO: 8, the light chain polypeptide of the amino acid sequence SEQ ID NO: 5 can be encoded, for example, by the polynucleotide sequence SEQ ID NO: 11.

[0044] Another preferred antibody contains a heavy chain polypeptide of the amino acid sequence SEQ ID NO: 4 and a light chain polypeptide of the amino acid sequence SEQ ID NO: 7. The heavy chain polypeptide of the amino acid sequence SEQ ID NO: 4 can be encoded, for example, by the polynucleotide of the sequence SEQ ID NO: 10, the light chain polypeptide of the amino acid sequence SEQ ID NO: 7 can be encoded, for example, by the polynucleotide of the sequence SEQ ID NO: 13.

[0045] Another preferred antibody contains a heavy chain polypeptide of the amino acid sequence SEQ ID NO: 3 and a light chain polypeptide of the amino acid sequence SEQ ID NO: 6. The heavy chain polypeptide of the amino acid sequence SEQ ID NO: 3 can be encoded by the polynucleotide of the sequence SEQ ID NO: 9, the light chain polypeptide of the amino acid sequence SEQ ID NO: 6 can be encoded by the polynucleotide of the sequence SEQ ID NO: 12.

[0046] The most preferred humanized antibodies are designated herein as 86, 88 and 89. The SEQ ID NOs of their sequences are listed in Table 1.

[0047] Preferably, an antibody in which all six CDRs, HCVR, LCVR, HCVR, and LCVR, the entire heavy chain, the entire light chain, or the entire heavy chain and the entire light chain are capped by a particular sequence, as shown herein in SEQ ID NO (see, e.g., Table 1), is characterized by having a KD for human sclerostin, at 25°C, of less than about 10 pM, 8 pM, 6 pM or 4 pM, preferably less than about 2.2 pM. In addition, it is preferred that such an antibody is further limited by having a macaque sclerostin Kdza at 25°C of less than about 100 pM, 90 pM or 80 pM, or more preferably less than about 75 pM.

[0048] Preferably, an antibody in which all six CDRs, HCVR, LCVR, HCVR, and LCVR, the entire heavy chain, the entire light chain, or the entire heavy chain and the entire light chain are capped by a particular sequence, as shown herein in SEQ ID NO, is characterized by having an IC50 in a bone-specific alkaline phosphatase assay with human sclerostin (see, e.g., Example 2 herein) of about 50 nM or less, about 40, 35, or 30 nM or less, more preferably about 25 nM, more preferably about 20 nM (eg, 20.2 nM) or less.

[0049] Even more preferably, an antibody in which all six CDRs, HCVR, LCVR, HCVR, and LCVR, the entire heavy chain, the entire light chain, or the entire heavy chain and the entire light chain are bounded by a particular sequence, as shown herein in SEQ ID NO, is characterized by having a Kdna of 25°C or less than about 10 pM in a bone-specific alkaline phosphatase assay with human sclerostin, 8 pM, 6 pM or 4 pM, preferably less than about 2.2 pM, and which is also characterized in that in a bone-specific alkaline phosphatase assay, with human sclerostin, it will have an IC of about 50 nM or less, about 40, 35, or 30 nM or less, more preferably about 25 nM or less, even more preferably about 20 nM (eg, 20.2 nM) or less.

[0050] Even more preferably, an antibody in which all six CDRs, HCVR, LCVR, HCVR, and LCVR, the entire heavy chain, the entire light chain, or the entire heavy chain and the entire light chain are capped by a particular sequence, as shown herein in SEQ ID NO, is characterized by having a Kdza of human sclerostin at 25°C of less than about 10 pM, 8 pM, 6 pM or 4 pM, preferably less than about 2.2 pM; also characterized by having an IC50u of a human sclerostin bone-specific alkaline phosphatase assay of about 50 nM or less, about 40, 35, or 30 nM or less, more preferably about 25 nM or less, even more preferably about 20 nM (eg, 20.2 nM) or less; and is also characterized by having an IC50 of about 75 nM or less in the macaque monkey sclerostin bone-specific alkaline phosphatase assay.

Antibody expression

[0051] The present invention also relates to host cells expressing the sclerostin antibody of the present invention. Generation and isolation of host cell lines. produced by the antibodies of the present invention can be performed by techniques known in the art.

[0052] A variety of host expression systems known in the art can be used to express an antibody of the present invention, including prokaryotic (bacterial) and eukaryotic expression systems (such as yeast, baculovirus, plants, mammalian and other animal cells, transgenic animals, and hybridoma cells), as well as phage display systems.

[0053] The antibody of the present invention can be obtained by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. For recombinant antibody expression, the host cell is transformed, transduced, infected, and si. by one or more recombinant expression vectors carrying DNA fragments encoding immunoglobulin light and/or heavy chains of antibodies so that the light and/or heavy chains are expressed in the host cell. The heavy chain and the light chain can be expressed independently from different promoters to which they are operably linked in one vector or, alternatively, the heavy chain and the light chain can be expressed independently from different promoters to which they are operably linked in two vectors - one expressing the heavy chain and one expressing the light chain. Optionally, the heavy chain and the light chain can be expressed in different host cells.

[0054] In addition, the recombinant expression vector may also encode a signal peptide that facilitates the secretion of light and/or heavy chain antibodies to sclerostin from host cells. The sclerostin antibody light and/or heavy chain gene can be cloned into a vector such that the signal peptide is operably linked in the same reading frame to the portion of the gene encoding the amino terminus of the antibody chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide. Preferably, the recombinant antibodies are secreted into the host cell culture medium from which the antibodies can be recovered or purified.

[0055] Isolated DNA encoding the HCVR region can be translated into a gene encoding the full-length heavy chain by operatively linking the DNA encoding HCVR to another DNA molecule encoding the heavy chain constant regions. The gene sequences for the heavy chain constant regions of humans and other mammals are known. DNA fragments surrounding these regions can be obtained, e.g., by standard PCR amplification. The heavy chain constant region can be a constant region of any type {eg, IgG, IgA, IgE, IgM or IgD), class {eg, IgGi, IgG2, IgG3, and IgG4) or subclass and any allotype variant thereof as described by Kabat {supra).A preferred heavy chain constant region comprises the polypeptide sequences of SEQ ID NO:38.

[0056] Isolated DNA encoding the LCVR region can be translated into a full-length light chain gene (as well as a light chain Fab gene) by operatively linking the DNA encoding the LCVR to another DNA molecule encoding the light chain constant region. Gene sequences encoding the constant region of humans, ko and other mammals are known in the art. DNA fragments surrounding these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0057] Most suitable mammalian host cells for use in the present invention are CHO cells {eg, ATCC CRL-9096), NSO cells, SP2/0 cells and COS cells (ATCCeg, CRL-1650, CRL-1651), HeLa (ATCC CCL-2). Additional host cells for use in the present invention include other mammalian cells, yeast cells, and prokaryotic cells. When recombinant expression vectors containing genes for antibodies are introduced into mammalian host cells, the antibodies are obtained by culturing the host cells for a period of time sufficient to express the antibodies in the host cells or, even better, to secrete the antibodies into the culture medium in which the host cells are grown. Antibodies can be obtained from host cells and/or culture media using standard purification methods.

[0058] Once expressed, complete antibodies, individual light and heavy chains, or other forms of immunoglobulins of this invention can be purified by standard procedures known in the art, including ammonium sulfate precipitation, column chromatography - ion exchange, affinity, reverse-phase, hydrophobic interactions, gel electrophoresis, and the like. Pure immunoglobulins of at least about 90%, 92%, 94% or 96% homogeneity, more preferably 98 to 99% or greater homogeneity, are preferred for pharmaceutical use. Once purified, partially or to the desired homogeneity, the sterile antibodies can be used therapeutically, as shown herein.

Humanized antibodies

[0059] It is desirable that the antibody of this invention to be used for therapeutic purposes has a framework and constant region sequence (to the extent that it exists in the antibody) derived from the mammal in which it would be applied as a therapeutic agent, to reduce the possibility that the mammal develops an immune response to the therapeutic antibody. Humanized antibodies are of particular interest as they are useful in therapeutic applications and reduce the likelihood of an immune response often seen with antibodies of murine origin or antibodies containing portions of murine origin when these are administered to humans. Predominantly, injected humanized antibodies can have a half-life more similar to natural human antibodies than, for example, murine antibodies, thus achieving lower and lower doses to the subject.

[0060] The term "humanized antibody", as used herein, refers to an antibody in which at least one part is of human origin. For example, a humanized antibody may contain portions derived from an antibody of non-human origin, such as a mouse, and portions derived from a human antibody joined, e.g., chemically, by conventional techniques (e.g., synthesis) or obtained as a continuous polypeptide, using genetic engineering techniques.

[0061] Preferably, the "humanized antibody" has CDRs originating from, or derived from, the parent antibody, i.e., from a non-human antibody (mainly a mouse monoclonal antibody), while the framework and constant region, to the extent that they are present (or a significant or essential part thereof, i.e., at least about 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99%) encodes the nucleic acid sequence information found in the human embryonic immunoglobulin region (see, e.g., the International ImMunoGeneTics Database) or in its recombined or mutated forms, regardless of whether said antibodies are produced in human cells. Preferably, at least two, three, four, five, or six CDRs of the humanized antibody are optimized from the CDRs of the non-human parent antibody from which the humanized antibody is derived, to produce a desired property, e.g., improved specificity, affinity, or neutralization, which can be determined by screening assays, e.g., ELISA. It is desirable that the optimized CDR in an antibody of the present invention contains at least one amino acid substitution in relation to the original antibody. Certain amino acid substitutions in the CDRs of the humanized antibody 88 and 89 of the present invention, compared to the parent antibodies 788 and 789 (see Examples 5 and 6 herein), reduce the likelihood of antibody instability (eg, removal of Asn residues from the CDR) or immunogenicity of the antibody when administered to a human (eg, as predicted by IMMUNOFILTER™ Technology).

[0062] Humanized antibodies predominantly contain minimal sequences derived from non-human antibodies. Humanized antibodies may contain amino acids that are not found in either the recipient antibody or the framework or CDR sequences derived from the parent antibody. Humanized antibodies can be subjected to in vitro mutagenesis using routine methods used in the art, and therefore the amino acid sequences of the HCVR and LCVR framework regions of humanized recombinant antibodies are sequences that, although derived from human embryonic HCVR and LCVR sequences, may not be found in the natural repertoire of human embryonic antibodies in vivo. It is believed that those amino acid sequences of the HCVR and LCVR framework regions of humanized recombinant antibodies are at least 90%, 92%, 94%, 95%, 96%, 98% or, more preferably, at least 99% or, most preferably, 100% identical to Human embryonic sequences.

[0063] In most preferred embodiments, the humanized antibody of the present invention comprises human light chain embryonic framework region sequences (see, e.g., PCT WO 2005/005604) and human heavy chain embryonic framework region sequences (see, e.g., PCT WO 2005/005604). The most preferred human embryonic light chain framework region sequences are from the human kappa light chain gene, selected from the group consisting of Al 1, A17, A18, A19, A20, A27, A30, LI, LI 1, L12, L2, L5, L6, L8, 012, 02, and 08. The most preferred human embryonic heavy chain framework region sequences are from the heavy chains human selected from the group consisting of: VH2-5, VH2-26, VH2-70, VH3-20, VH3-72, VH1-24, VH1-46, VH3-9, VH3-66, VH3-74, VH4-31, VH1-18, VH1-69, VH3-7, VH3-11, VH3-15, VH3-21, VH3-23, VH3-30, VH3-48, VH4-39, VH4-59, VH5-51 (see, International Publication No. WO2006/046935).

[0064] There are many methods in the art for obtaining humanized antibodies. For example, humanized antibodies can be produced by obtaining the nucleic acid sequences encoding the HCVR and LCVR of a parent antibody (eg, mouse antibody or hybridoma-forming antibody) that specifically binds sclerostin, preferably human sclerostin, identifying the CDRs in those HCVRs and LCVRs (non-human), and inserting the nucleic acid sequences encoding such CDRs into selected sequences encoding human framework regions. Optionally, the CDR region can be optimized by random mutagenesis or mutating specific positions to replace the DNA or multiple amino acids in the CDR with a different amino acid before the CDR region is transferred to the framework region. Alternatively, the CDR region can be optimized after insertion into the human framework region by methods available to those skilled in the art.

[0065] As the CDR encoding sequences are transferred to selected human framework region sequences, the resulting DNA sequences encoding the humanized heavy variable region and humanized light chain variable region sequences are then expressed to yield a humanized sclerostin-binding antibody. Humanized HCVR and LCVR can be expressed as part of a whole sclerostin antibody, ie, as a fusion protein with human constant domain sequences. However, the HCVR and LCVR sequences can also be expressed without the constant sequences to give a humanized anti-sclerostin Fv.

[0066] References further describing methods that can be used to humanize mouse antibodies include, e.g., Queen et al., Proc. Natl. Acad. Sci. USA 88:2869, 1991 and the method of Winter et al. [Jones et al., Nature, 321:522 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeven et al., Science, 239:1534 (1988)].

Diagnostic applications

[0067] An antibody of the present invention can be used for the diagnosis of a disorder or disease related to the expression of human sclerostin. Similarly, an antibody of the present invention can be used to control sclerostin levels in a subject being treated for a sclerostin-related condition. These applications include methods that use an antibody of the invention and label sclerostin in a biological sample, eg, in a human body fluid or in a cell or tissue extract (see, eg, Example 1 herein). Antibodies of the present invention may be used with or without modifications and may be labeled by covalent or non-covalent attachment of a detectable group.

[0068] A number of conventional protocols for measuring the level of sclerostin in a biological sample are known in the art, including e.g., ELISA, RIA, and FACS, which provide a basis for diagnosing altered or abnormal levels of sclerostin expression. Normal or standard levels of sclerostin present in a sample are established using any known technique, eg, by combining a sample containing a sclerostin polypeptide with, eg, an antibody of the present invention under conditions suitable for antibody-antigen complexation. The antibody is directly or indirectly labeled with a detectable substance to facilitate detection of bound or free antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances. The amount of the formed standard complex is quantified by different methods, such as, for example, photometrically. The amount of sclerostin polypeptide present in the samples is then compared with standard values.

Therapeutic<p>Rings

[0069] Sclerostin functions as a negative regulator of bone formation, (see, e.g., Cytokine & Growth Factor Review, 16:319-327, 2005). In adults, sclerostin mRNA is primarily detected in osteocytes, although applicants have found lower concentrations in cartilage.

[0070] A pharmaceutical composition containing a monoclonal antibody to sclerostin of the present invention can be used to increase at least one of the following: bone mass, bone mineral density, bone mineral content, or bone strength, either in the bones of the spine, or in other bones, or both, when an effective amount is administered to a person in need thereof. A pharmaceutical composition comprising a monoclonal antibody to sclerostin of the present invention can be used to reduce the incidence of fractures of the spine or other bones when an effective amount is administered to a person in need thereof. Reducing the incidence of fractures involves reducing the probability or actual incidence of fractures in that individual compared to the untreated population.

[0071] In addition, the antibody of the present invention may be useful for treating conditions, diseases, or disorders in which the presence of sclerostin causes or contributes to unwanted pathological effects or where reduction of sclerostin levels or sclerostin bioactivity provides therapeutic benefit to certain individuals. Such conditions, diseases or disorders include, but are not limited to, osteoporosis, osteopenia, osteoarthritis, pain related to osteoarthritis, periodontal disease, or multiple myeloma. Persons can be male or female. Generally, people are at risk of breaking their spine or other bones, more often in people who are at risk of or already suffer from osteoporosis. More often, people are female, and even more often women at risk or who already have post-menopausal osteoporosis. It is believed that the method of the present invention can benefit a person at any stage of osteoporosis.

[0072] In addition, the use of the antibodies of the present invention for the manufacture of a medicament for the treatment of at least one of the aforementioned disorders is contemplated.

[0073] The terms "treatment" and "treating" refer to all processes in which there may be slowing, interruption, stoppage, control or stopping of the progression of the disorders mentioned here, but does not necessarily indicate the complete removal of all symptoms of the disorder. "Treatment", as used herein, includes the administration of a compound of the invention to treat a disease or condition in a mammal, particularly a human, and includes: (a) inhibiting further progression of the disease, i.e., halting its development, and (b) attenuating the disease, i.e., causing regression of the disease or disorder or alleviation of symptoms or complications. Dosing regimens can be adjusted to provide the optimum desired response (eg, therapeutic response). For example, only one bolus can be given, several divided doses over time, or the dose can be proportionally reduced or increased depending on the requirements of the therapeutic situation.

Pharmaceutical composition

[0074] The antibody of the present invention can be incorporated into a pharmaceutical composition suitable for human administration. An antibody of the present invention may be administered to a subject alone, or in combination with a pharmaceutically acceptable carrier and/or diluent in one or more doses. Such pharmaceutical compositions are made to correspond to the selected method of administration, and pharmaceutically acceptable solvents, diluents, carriers and/or excipients, such as dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents and the like. are used as appropriate. Said compositions may be designed in accordance with conventional techniques described in, e.g., Remington, The Science and Practice of Pharmacv, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA 1995, which provide a concise overview of formulation techniques and are generally known to those skilled in the art. Suitable carriers for pharmaceutical compositions include any materials which, when combined with a monoclonal antibody of the present invention, retain the activity of the molecule and do not interact with the immune system of the individual to whom it is administered.

[0075] A pharmaceutical composition comprising a monoclonal antibody to sclerostin of the present invention can be administered to a person at risk of or already exhibiting the pathologies described herein, eg, osteoporosis, osteoarthritis or other degenerative bone disorders, using standard administration techniques.

[0076] The pharmaceutical composition of the present invention generally contains an "effective amount" of the antibody of the present invention. Effective amount refers to the amount needed to (at doses, and for the period of time and for the method of administration) achieve the desired therapeutic result. The effective amount of antibody may vary due to factors such as the disease state, age, sex, and weight of the individual and the ability of the antibody or antibody portion to elicit the desired response in the individual. An effective amount is also that amount at which any toxic or harmful effects of the antibody outweigh the therapeutic beneficial effects.

[0077] An effective amount is at least the minimum dose, but less than the toxic dose, of an active agent necessary to provide therapeutic benefit to a subject. In other words, an effective amount or a therapeutically effective amount of an antibody of this invention is an amount that in mammals, primarily humans, (i) increases at least one of the following: bone mass, bone mineral density, bone mineral content or bone strength or (ii) treats a condition, disorder or disease where the presence of sclerostin causes or leads to an unwanted pathological effect, or (iii) reduction of sclerostin or sclerostin bioactivity leads to a beneficial therapeutic effect in mammals, preferably a human, including but not limited to osteoporosis, osteopenia, osteoarthritis, rheumatoid arthritis, periodontal disease or multiple myeloma.

[0078] As is well known in the medical art, the dosage for each individual subject depends on many factors, including the patient's size, body surface area, age, compound being administered, sex, time and route of administration, general medical condition, and other concomitant medications. Furthermore, the dose will vary depending on the type and severity of the disease. A typical dosage may be, for example, in the range of 0.001 to 1000 µg; better from 1 to 100 ug; However, lower and higher doses than those mentioned are also foreseen, especially when the mentioned factors are taken into account. A daily parenteral dosage regimen may be about 0.1 µg/kg to about 20 mg/kg of total body weight, preferably from about 0.3 µg/kg to about 10 mg/kg. Progress can be monitored with periodic checks and the dose adjusted accordingly.

[0079] These suggested amounts of antibody are subject to a large degree of therapeutic discretion. The key factor in choosing the appropriate dose and schedule is the result obtained. Factors considered in this context include the disorder being treated, the clinical condition of the individual patient, the cause of the disorder, the site of antibody delivery, the type of antibody given, the method of antibody administration, and other factors known to the medical practitioner.

[0080] The method of administration of the antibody<*> of this invention may be oral, parenteral, by inhalation, or topically. Preferably, the antibodies of the present invention are incorporated into a pharmaceutical composition suitable for parenteral administration. The term parenteral, as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, or intraperitoneal administration. Parenteral administration by intravenous or intraperitoneal or subcutaneous injection is preferred. Subcutaneous injections are preferred. Suitable vehicles for such injections are well known in the art.

[0081] Typically, the pharmaceutical composition must be sterile and stable under the conditions of manufacture and storage in its package, which may be, e.g., a sealed vial, syringe or other administration device, e.g., a "pen". Therefore, pharmaceutical compositions can be sterilized by filtration after the formulation is made, or made microbiologically acceptable in some other way.

[0082] The following examples are provided for illustration only and are not intended to limit the scope of the present invention.

EXAMPLES

Example 1 ELISA test

[0083] This assay is used for the detection and quantification of sclerostin in human serum samples down to a lower detection limit of 0.4 ng/ml. Antibody 86 is a binding antibody (capture antibody), while antibody 88 is a detection antibody. (see Table 1 for antibody sequences).

[0084] The inside of the wells of a 96-well plate were coated with 100 µl each of whole monoclonal antibodies to sclerostin 86, at a concentration of 0.5 µg/mL in 0.5 M sodium carbonate pH 9.6 ("coating buffer"). The plate was warmed and incubated overnight at 4°C. The plate was then washed three times with TBST "wash buffer" (0.4 M Tris-HCl, 3 M NaCl, 0.1% Tween 20). Then 200 µL of casein blocking buffer (blocking buffer) in PBS (Pierce, #37528) was added to each well and the plate was incubated for one hour at room temperature. The plate was then washed twice with wash buffer to remove the stop buffer.

[0085] To obtain a standard curve, human sclerostin at a concentration of 0.35 mg/mL was prepared in casein stop buffer and serially diluted twice (from 1.25 ng/ml to 0 ng/ml) with 5% pooled human serum in casein stop buffer in PBS. Human serum was passed through a column of magnetic beads coated with a monoclonal antibody to sclerostin 86, to remove any sclerostin that might be endogenously present in the serum.

[0086] One hundred microliters of sample (5% serum in casein stop buffer) or standard was added in duplicate to the wells and the plate was additionally incubated at room temperature for two hours. The plates were then washed 5 times with wash buffer.

[0087] One hundred microliters of detection antibody (monoclonal antibody to full-length sclerostin, no. 88) that was biotinylated using the Pierce EZ link NHS-LC biotin kit was added to each well. The plate was incubated at room temperature for one hour and then washed four times with washing buffer. Streptavidin-horseradish peroxidase was diluted 1:2000 with casein stop buffer in PBS to a final concentration of 0.5 ng/ml, and 100 µL was added to each well, incubated at room temperature for one hour and washed 7 times with wash buffer. OPD substrate (Sigma P8806), 100 µL/well, was added and the reaction was allowed to proceed for 20 minutes at room temperature. The reaction was then stopped by adding 100 µL/well of 1 N HCl. The plate was read at OD 490 nm.

Example 2 Neutralization - test with bone-specific alkaline phosphatase

[0088] Bone-specific alkama phosphatase is a biochemical indicator of osteoblast activity. The bone-specific alkaline phosphatase assay described here is based on the ability of sclerostin to reduce BMP-4 and Wnt3a-stimulated alkaline phosphatase levels in the mouse multipotent cell line C2C12, whereas, in this assay, an antibody neutralizing sclerostin activity would lead to a dose-dependent increase in alkaline phosphatase activity. Wnt3a and BMP-4 are osteogenic growth factors.

[0089] C2C12 cells (ATCC, CRL 1772) were seeded in a 96-well tissue culture plate, 3000-5000 cells per well, in MEM medium supplemented with 5% fetal bovine serum, then incubated overnight at 37°C in 5% CO2. The antibody to be tested was diluted in 0.5X Wnt3a-conditioned medium to various final concentrations. Then, the medium was removed from the cells in the tissue culture plate and 150 µl of previously prepared antibody-BMP4-sclerostin solution (human or macaque monkey) was added to each well, in which the antibody was at a final concentration of 30 ug/ml to 0.5 ng/ml, BMP-4 was at a final concentration of 25 ng/ml and sclerostin protein was at a final concentration of 1.0 ug/ml, and the conditioned medium was in a concentration of 0.5X. The plate was then incubated at 37°C in 5% CO2 for 72 hours. The medium was removed from the cells in the tissue culture plate, the cells were washed once with PBS, then the cell plate was frozen and thawed three times, alternately at -80°C and 37°C. ]0090] Conditioned medium with Wnt3a was obtained by growing L-Wnt-3A cells and control L-cells (ATCC CRL-2647, respectively CRL-2648) for four days, from 1:20 split confluent cells, in DMEM with 10% FBS and 2 raM L-glutamine. The collected medium was filtered through 0.2 micron nylon membranes and stored for a long time at -20°C or at 4°C for a short time.

[0091] Alkaline phosphatase activity is measured by adding 150 µl per well of alkaline phosphatase substrate (1-step PNPP, Pierce #37621). The cell plate was then incubated for 60 minutes at room temperature, when the absorbance, OD at 405 nm was read to determine alkaline phosphatase activity. The IC50 values for antibody neutralization given in Table 2 are the mean values from two separate experiments. IC50 calculations were performed using the SigmaPlot Regression Wizard, a 4-parameter sigmoid curve fitting DNA tool.

Example 3 Binding affinity

[0092] Equilibrium binding studies between antibodies to sclerostin and sclerostin of human, macaque ("cyno"" i.e., cyno, from Engl. cvnomolgous) monkeys or rats were performed on a KinExA 3000 instrument (Sapidvne Instruments Inc.) under Co-controlled conditions. With this technique, a fixed concentration of antibody, less than its Kd, is mixed with various concentrations of sclerostin protein and allowed to equilibrate. The fraction of free, unbound antibody that remains in solution is checked by briefly exposing this equilibrated mixture to sclerostin-coated beads, followed by detection with a fluorescently labeled secondary antibody. Typically, 2 pM of the test antibody to sclerostin is mixed with various concentrations of human, cyno, or rat sclerostin, starting at 2-50 nM and serially diluted threefold with binding buffer (IX soni phosphate buffered at pH 7.4, 0.02% sodium azide and 1 mg/mL bovine serum albumin) containing 2 pM of the test antibody. Allow these solutions to equilibrate for two days at 25°C. The amount of unbound antibody is checked using azlactone beads coated with human sclerostin (Sapidvine Instruments Inc.). These beads are prepared by reacting 50 mg of dry azlactone beads overnight, with rotation, with 50 mcg/ml human sclerostin in 50 mM sodium carbonate buffer pH 9.0-9.6. The beads are then allowed to settle, the supernatant is replaced with stop buffer (1 M Tris, pH 8.0, plus 10 mg/mL bovine serum albumin) and rotated for one hour. These beads are diluted 20-fold with binding buffer and used within three days. KinExA 3000 instrument, equilibrated at room temperature, (approx 25°C) was used for binding tests. Typically, 6.25 mL of the antibody/sclerostin equilibrated solution is passed through a packed column of human sclerostin-azlactone beads, at a flow rate of 0.25 mL/minute, then washed with buffer (IX soni phosphate-buffered saline at pH 7.4 with 0.02% sodium azide). The fraction of free sclerostin antibody bound to these beads is quantified by measuring the fluorescence obtained when injects a labeled secondary antibody. (Cy5-labeled goat anti-human Fab'2 antibody). For each condition, two samples were analyzed and KD was determined by fitting the data to a 1:1 binding model, using KinExA Pro software. The mean values of Kdna at 25°C are given in Table 3.

Example 4 invivocalcin test:

[0093] The calcein test on the rat enables immediate evaluation of bone formation in a short (10 days) period of time and the influence of an antibody of this invention on bone formation. Calcein is a fluorochrome marker that is incorporated into the bone matrix during new bone formation. Quantitative measurement of calcein monitors the degree of bone formation induced by pharmacological agents. Female Sprague-Dawley rats, 6 months old, were used (groups of n=6/dose). Rats were dosed every third day by subcutaneous injection (days 0, 3, and 6) with 1 mg/kg or 6 mg/kg antibody in PBS vehicle, or with 10 ng/kg PTH daily or with 6 mg/kg human IgG as a negative control. Calcein is injected subcutaneously on day 7 and rats are sacrificed on day 10. Tibiae were harvested and sectioned to assess trabecular bone formation (distal metaphysis) or cortical bone formation (tibial diaphysis), subjected to demineralization and fluorochrome quantitation of total calcein. The values shown in Table 4 represent the mean values when the human IgG control was taken as 1.0. These data show that administration of the antibodies of the invention lead to both trabecular and cortical bone formation.

Example 5Osteoarthritis

[0094] SOST expression is mostly restricted to bone tissue. However, Applicants herein determine that another tissue that exhibits significant expression of SOST, as measured by real-time PCR, is cartilage. SOST expression was also found with total RNA isolated from cartilage of patients with and without osteoarthritis. Furthermore, in total RNA, the expression of SOST increases in proportion to the severity of osteoarthritis, so that in the cartilage of mild osteoarthritis it is higher than in the control, in severe osteoarthritis it is higher than in mild, and in surgically severe cartilage (removed during knee replacement surgery) it is higher than in all others, which shows the correlation between osteoarthritis and SOST expression. A specific antibody to sclerostin can be used to treat osteoarthritis.

[0095] A chimeric antibody to sclerostin, with a mouse variable region in the rat Fc region, antibody 789 of the sequence SEQ ID NO:42 for the heavy chain and SEQ ID NO:43 for the light chain, was tested for its ability to stop joint pain in the MIA model of osteoarthritis. The variable region of this antibody was the DNA sequence for generating humanized antibody 89 (HCVR, sequence SEQ ID NO: 16 and LCVR, sequence SEQ ID NO: 19). Both antibodies, humanized antibody 89 and chimeric antibody 789, bind to the same epitope. This osteoarthritis model uses injections of monoiodoacetate (MIA) directly into the knee joint to induce an osteoarthritis-like process of cytokine-mediated inflammation and pain, as well as cartilage destruction. Soni solution alone is injected into the contralateral knee of rats and pain is measured as the difference in load bearing of the two hind legs.

[0096] In MIA experiment #1, young male Lewis strains (7-8 weeks of age) were used in a "preventive modality" in which the sclerostin antibody was administered prior to MIA injection. The day before MIA injection, rats were injected with either 10 mg/kg sclerostin antibody, i.p. or with 10 mg/kg control IgG, or PBS (n = 6 per group). The next day, the rats were anesthetized and their right knee was injected with 0.3 mg of monosodium iodoacetate dissolved in 50 µl of 0.9% sterile saline solution. Only the saline solution was injected into their left knee. Injection was performed through the patellar ligament using an insulin syringe with a 28G needle covered with plastic tubing to prevent needle penetration deeper than 5 mm into the knee joint.

[0097] Pain measurements were performed 2 and 7 days after MIA injection (3 and 8 days after antibody injection). After pain measurement on day 7, a second dose of antibody or a control injection was given. Repeat pain measurements were performed 10 and 14 days after MIA (3 and 7 days after the second antibody injection).

[0098] Pain was measured by the difference in the weight that the injected legs could bear, as measured by a weight-bearing test device. Rats were placed in Perspex cages with their hind paws on a pressure sensor. When the rats were completely still, measurements were taken for 1 second, after which two more measurements were taken and the mean value was calculated. The weight placed on the MIA-injected leg is subtracted from the weight placed on the saline-injected leg to give the difference in weight bearing.

[0099] A statistically significant reduction in pain was seen with the sclerostin antibody, relative to the IgG (and PBS) control at days 7, 10 and 14 after MIA administration (Table 5). Values are differences in submitted weights between the MIA-injected leg and the saline-injected leg, expressed in grams (DNA standard error of the mean).

[0100] The second MIA experiment was performed with older rats (27 weeks of age) in the "treatment modality", when the antibody is given after the injection of MIA. Rats received injections of MIA or control saline as before, and then, after 6 days, injected i.p. with 10 mg/kg of sclerostin antibody, control IgG, or PBS (n = 6 per group). On the 8th and 15th day after MIA (2 and 9 days after the antibody), pain measurements are taken as before. On day 16 post-MIA, a second dose of sclerostin antibody and controls were given, and on day 21 (5 days after the second antibody dose) pain measurements were again performed.

[0101] At early time points of sclerostin antibody administration (day 8 post-MIA), there is no effect of the antibody on pain, but 7 days later, there is a tendency towards pain reduction, as measured by the difference in weight bearing of the MIA-injected leg and the saline-injected leg, expressed in grams. The tendency towards pain reduction with the sclerostin antibody is also seen on the 5th day after the second dose of the antibody. Humanized antibody 789 differs from the IgG control by p-values of 0.06 and 0.08 at day 15 and 21, respectively. Taken together, these results demonstrate that pain resulting from an established MIA disease process is interrupted by administration of a neutralizing antibody to sclerostin.

Example 6 OVX studies in rats

[0102] The ovariectomized (OVX) rat is a well-known model for postmenopausal osteoporosis. In this study, the effects in the OVX rat of the sclerostin antibodies of the present invention, which contain the variable region of mouse and rat Fc.

[0103] Female Sprague-Dawley rats, 6 months old, were ovariectomized and allowed to lose bone for 1 month until dosing. JeDNA group of rats (n=8) were not ovariectomized, but sham operated to compare bones with ovariectomized rats. All OVX rats were randomly divided into different treatment groups (n=7 in each group) and treated with either PTH (1-38), or control with IgG (10 mg/kg) or 10 mg/kg chimeric antibody to sclerostin (antibody 788 with the heavy chain of SEQ ID NO: 40 and the light chain of SEQ ID NO: 41 [the variable region of this antibody was the resulting DNA sequence for generation of humanized antibody 88]; antibody 789 with the heavy chain of the sequence SEQ ID NO: 42 and the light chain of the sequence SEQ ID NO: 43), a total of 58 days. All antibodies were administered every 3rd day subcutaneously, while PTH (1-38) was administered daily subcutaneously, 10 mg/kg. After sacrifice, femurs and vertebrae were removed for quantitative computed tomography (CT) analysis using a CT scanner of the distal femur (trabecular bone) and midshaft (midshaft, cortical bone), as well as the 5th lumbar vertebra. The bones are placed on molded clay to obtain reproducible measurements and scans are taken of the femur 2 mm from the end of the epiphysis (distal femur) or 10 mm from the epiphysis (mid femur).

[0104] Vertebral measurements were taken with L-5, scanned from the "V" sign in the vertebral structure. Data were calculated using the commercial software package SYS-C320-V 1.5. Biomechanical properties of the femoral diaphysis, L-5 vertebra, and femoral neck were measured post-mortem, according to: Turner CH, Burr DB, Basic biomechanical measurements of bone: a tutorial. Bone 14(4):595-608, 1993. Midsection mechanical properties were measured for the intact left femur using 3-point bending with a load placed between two supports 15 mm apart. The femurs are positioned so that the load point is approximately 7.5 mm from the lower leg and bending occurs around the medial-lateral axis. The samples were tested in a sodium solution bath at 37°C. Load-displacement curves were recorded at a speed of 10 mm/min using a material testing apparatus (model: l/S, MTS Corp, Minneapolis, MN) and analyzed with TestWorks 4 software (MTS Corp.) to calculate the maximum load. The mechanical properties of the L-5 vertebrae were analyzed after the posterior processes were removed and the ends of the centrum brought into a parallel position using a diamond saw (Buehler Isomet, Evanston, IL). Vertebral specimens were loaded in compression using a material testing machine and analyzed with TestWorks 4 software (MTS Corp.). For femoral neck measurements, the femur was placed upright in the drill head with the proximal side up, and the load was applied downward, in the center of the femoral head. Analysis was performed with TestWorks 4 software (MTS Corp.). Means and standard errors of the means are listed in Tables 5 and 6. The data show that antibody chimeras 788 and 789 increase bone mineral density (BMD), bone mineral content (BMC), and bone strength (peak load) in OVX rats.

LIST OF SEQUENCES

[0105]

Claims (8)

1. An antibody or its functional fragment that specifically binds human sclerostin, where the antibody or its functional fragment contains six CDRs of the following amino acid sequences: HCDR1 sequence SEQ ID NO: 26, HCDR2 sequence SEQ ID NO: 27, HCDR3 sequence SEQ ID NO: 28, LCDR1 sequence SEQ ID NO: 29, LCDR2 sequence SEQ ID NO: 30 and LCDR3 sequence SEQ ID NO: 31. 2. The antibody or its functional fragment according to claim 1, which contains a heavy chain variable region and a light chain variable region, where the heavy chain variable region has the amino acid sequence of SEQ ID NO: 15, and the light chain variable region has the amino acid sequence of SEQ ID NO: 18. 3. The antibody or its functional fragment according to claim 1 or according to claim 2, which contains a heavy chain polypeptide of the amino acid sequence SEQ ID NO: 3 and a light chain polypeptide of the amino acid sequence SEQ ID NO: 6. 4. A pharmaceutical composition comprising the antibody of any of the preceding claims and a pharmaceutically acceptable carrier or diluent. 5. The antibody or functional fragment thereof according to any one of claims 1 to 3 for use as a medicine. 6. The antibody or functional fragment thereof according to any one of claims 1 to 3 for use in increasing at least one of the following: bone mass, bone mineral density, bone mineral content or bone strength in humans. 7. The antibody or functional fragment thereof according to any one of claims 1 to 3 for use in the treatment of diseases or disorders such as osteoporosis, osteopenia, osteoarthritis, pain associated with osteoarthritis, rheumatoid arthritis, periodontal disease, or multiple myeloma in humans. 8. The antibody or its functional fragment according to claim 7 for use in the treatment of osteoporosis.